Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily

• Published in: Cell Vol. 145; no. 2; pp. 198 - 211
• Main Authors: Tsutakawa, Susan E., Classen, Scott, Chapados, Brian R., Arvai, Andrew S., Finger, L. David, Guenther, Grant, Tomlinson, Christopher G., Thompson, Peter, Sarker, Altaf H., Shen, Binghui, Cooper, Priscilla K., Grasby, Jane A., Tainer, John A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2011
• Subjects: active sites; Amino Acid Sequence; base pair mismatch; Catalytic Domain; DNA - metabolism; DNA Mutational Analysis; DNA repair; DNA replication; Exodeoxyribonucleases - chemistry; Exodeoxyribonucleases - metabolism; Flap Endonucleases - chemistry; Flap Endonucleases - metabolism; homologous recombination; Humans; Models, Molecular; Molecular Sequence Data; oligonucleotides; phosphates; RNA; Sequence Alignment; single-stranded DNA; Substrate Specificity
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2011.03.004

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5′ flaps. FEN1 5′ nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3′ and 5′ flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5′ ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity. [Display omitted] ► Structures and mutations of FEN1:DNA complexes reveal 5′ flap recognition mechanism ► FEN1 binds 100° bent DNA and unpaired 3′ flap, threading the 5′ end for specific incision ► FEN1 disorder-to-order transition on substrate DNA binding aligns active site ► Two nucleotides of the 5′ flap must unpair to productively position DNA in active site